The present invention relates to therapeutic protocols and pharmaceutical compositions designed to target Src family kinases and components of the Src kinase family signal transduction pathways, including HBx activation of Src kinase family signal transduction pathways for the treatment and prevention of hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC). The invention also relates to screening assays to identify potential antiviral agents which target HBx-mediated activation of Src kinase signaling cascades for the treatment of HBV.
Infection with HBV is an international public health problem of wide proportions. It has been estimated that at least 10% of the population of tropical Africa and Far-East Asia are chronic carriers of the virus (Tiollais et al., 1985, Nature 317:489-495). HBV is a hepatotropic virus whose course of infection can range from inapparent to acute hepatitis and severe chronic liver disease (Tiollais et al., 1985, Nature 317:489-495). Epidemiological studies have estimated that 250 million people are chronic carriers of HBV and serve as a reservoir for continued infections. Although the mechanism remains obscure, these HBV carriers have more than a 200 fold greater risk for development of hepatocellular carcinoma (HCC) (Beasley et al., 1981, Lancet 2:1129-1133).
HBV is a DNA-containing para-retrovirus that replicates by reverse transcription but comprises a separate family of viruses from retroviruses, known as hepadnaviruses. Human HBV is the prototype virus in a family that all possess a similar viral architecture and genetic arrangement, although only infection with the mammalian hepadnaviruses HBV (Tiollais et al., 1985, supra), woodchuck hepatitis B virus (WHV) (Popper et al., 1987, Proc. Natl. Acad. Sci. 84:866-870), and possibly ground squirrel hepatitis B virus (GSHV) (Marion et al., 1986, Proc. Natl. Acad. Sci. 83:4543-4546; Seeger et al., 1991, J. Virol. 65:1673-1679) cause both acute and chronic active hepatitis and HCC.
Acute hepatitis following a primary infection with HBV is usually self-limited in adults and often asymptomatic. Following acute hepatitis, 80-90% of infected adult individuals will clear viral antigens from liver and blood, resulting in clinical recovery and immunity to reinfection (Kumar et al., 1992, Basic Pathology, Fifth Edition (Philadelphia: W.B. Saunders Company)). However, 5-10% of individuals do not resolve the primary infection, instead developing a persistent hepatic infection (Ganem and Varmus, 1987, Ann. Rev. Biochem. 56:651-693). Chronic carriers represent a minority outcome following HBV infection, but constitute the majority of cases of HBV-related morbidity and mortality. Infection of infant and newborns results in a high carrier rate (approximately 90%), in contrast to infection of adults. Chronic carriers serve as the reservoir from which HBV is spread both horizontally (through blood and sexual contact) and vertically (from carrier mothers to newborns). Furthermore, chronic HBV infection frequently results in premature death from hepatic cirrhosis and liver failure (Ganem et al., 1987, Ann. Rev. Biochem. 56: 651-693). As previously noted, chronic carriers have a more than 200 fold increased risk for development of primary hepatocellular carcinoma (Beasley et al., 1981, Lancet 2:1129-1133). Because infection by HBV strongly correlates with development of HCC, considerable effort has been expended in identifying potential mechanisms for tumorigenicity by HBV (reviewed in Ganem et al. 1987, supra; Robinson, 1994, Ann. Rev. Med. 45:297-323; Rogler, 1991, Curr. Top. Micro. Immunol. 168:103-140). However, no clear mechanism has been described for the association between HCC and infection with HBV.
There are currently very limited therapeutics available for the treatment of HBV infection. Anti-HBV vaccines are currently being used to prevent HBV infection. However, the efficacy of these vaccines to treat chronic HBV infection and the availability of these vaccines to treat this worldwide health problem remains to be determined. Therefore, the need for an effective anti-HBV therapeutic still exists today.
The HBx protein is encoded by one of the four conserved open reading frames of the HBV genome. The L(+) (coding) strand encodes the four conserved open reading frames (ORFs) and codes for all the viral proteins (Ganem et al., 1987, supra). Four mRNAs have been identified. A 2.4 kb preS1 mRNA encodes the large surface antigen (pre-S1) and a 2.1 kb preS2/S mRNA encode the middle (pre-52) and small (major; S) surface antigens (Tiollais et al., 1985, supra). The 3.4 kb pregenome mRNA encodes the precore and core proteins, as well as the polymerase (P). The core protein is the principal structural component of the viral nucleocapsid and possesses nucleotide binding activity. The P protein, which has RNaseH activity, is the viral reverse transcriptase and the protein primer for synthesis of the L(xe2x88x92) strand (Robinson, 1994, Ann. Rev. Med. 45:297-323). The fourth mRNA is xcx9c0.7 kb in size, and is thought to encode the transcriptional transactivator known as HBx. HBx is a conserved 154 amino acid polypeptide which corresponds to a protein of a molecular weight of xcx9c17 kilodaltons.
The HBx protein is highly conserved within different mammalian HBV serotypes. However, in contrast to the other viral polypeptides, the role of HBx in the HBV life cycle is not yet understood. HBV-infected patient sera indicate that anti-HBx antibodies are produced (Elfassi et al., 1986, Proc. Natl. Acad. Sci. 83:2219-2222; Meyers et al., 1986, J. Virol. 57:101-109), demonstrating that expression of HBx does occur at some stage of HBV infection. HBx protein has also been detected in the livers of patients with chronic hepatitis (Haruna et al., 1991, Hepatol 13:417-421; Katayama et al., 1989, Gastroenterology 97:990-998). Patients testing positive for HBx expression have been found to have increased serum levels of HBV, thereby correlating HBx expression with increased viral replication (Haruna et al., 1991, Hepatol 13:417-421).
The precise role for HBx in the viral infectious process and in the development of HCC remains obscure. There are conflicting reports as to the role of HBx in the viral infectious process and in the development of HCC. It has been reported that there is a correlation between high levels of HBx expression and the development of HCC in transgenic mice. (Kim et al., 1991, Nature 353:317-320; Koike et al., 1994, Hepatol 19:810-819). However, these results remain controversial, as other groups have found no significant liver disease in HBx expressing mice (Balsano et al., 1994, J. Hepatol. 21:103-109; Dandri et al., 1996, J. Virol. 70; Lee et al., 1990, J. Virol. 64:5939-5947).
Several groups have shown HBx to be a largely if not entirely cytoplasmic protein, although 5-10% of HBx may reside in the nucleus (Doria et al. 1995, EMBO J. 14:4747-4757; Dandri et al. 1996 J. Virol. 70). HBx cannot be found to measurably associate with organelles, membrane vesicles or intermediate filaments, although some preferential accumulation near the cell surface can be observed (Doria et al., 1995, EMBO J. 14:4747-4757). HBx is a weak to moderately strong transcriptional transactivator. HBx has been shown to transactivate transcription of the interferon-xcex2 gene (Twu et al., 1987, J. Virol. 61:3448-3453) and of the HBV enhancer (Spandau et al., 1988, J. Virol. 62:427-434). Since those first reports, HBx has been shown to transactivate a wide variety of cellular and viral transcriptional elements (reviewed in Yen, 1996, J. Biomed. Sci. 3:20-30). Activation has been localized to specific binding sites for the transcription factors AP-1 (Benn and Schneider, 1994, Proc. Natl. Acad. Sci. 91; Natoli et al., 1994, Mol. Cell. Biol. 14:989-998; Seto et al., 1990, Nature 344:72-74), AP-2 (Seto et al., 1990, supra), NF-xcexaB (Lucito and Schneider, 1992, J. Virol. 66:983-991; Mahe et al., 1991, J. Biol. Chem. 266:13759-13763; Su and Schneider, 1996 J. Virol. 70:4558-4566; Twu et al., 1989, J Virol. 61:3448-3453), ATF/CREB (Maguire et al., 1991, Science 252:842-844) and possibly c/EBP (Faktor and Shaul, 1990; Mahe et al., 1991, supra).
HBx does not contain any structural motifs that convincingly suggest a function, such as DNA binding (Lucito and Schneider, 1993 in Animal Viruses, L. Carrasco ed. (NY: Plenum Press) p. 67-80), nor has it been observed to directly bind DNA (Siddiqui et al., 1987, Virol. 169:479-484; Wu et al., 1990 Proc. Natl. Acad. Sci. USA 84:2678-2682). A number of activities have been ascribed to HBx including an in vitro association with p53 (Butel et al., 1996, Trend Micro. 4:119-124), an association with the human homolog of a UV-damage DNA repair protein (Lee et al., 1995, J. Virol. 69:1107-1174), and an association with a serine protease inhibitory protein (Takada et al., 1994, Oncogene 9:341-348). In summary, it appears that the activities of HBx are not limited solely to transcriptional transactivation, and surely other HBx-associated activities will be discovered.
One early model suggested that HBx indirectly stimulates transcription through activation of a protein kinase C (PKC) signaling pathway (Kekule et al., 1993, Nature 361:742-745). Many groups report PKC-independent transactivation by HBx (Benn et al., 1996, J. Virol. 70:4978-4985; Chirillo et al., 1995, J. Virol. 70; Cross et al., 1993, Proc. Natl. Acad. Sci. 90:8078-8082; Lucito and Schneider, 1992, supra; Murakami et al., 1994, Virol. 199:243-246; Natoli et al., 1994, supra). It was demonstrated that HBx activation of AP-1 and NF-xcexaB factors occurs by HBx activation of a Ras signal transduction cascade (Benn and Schneider, 1994 supra; Cross et al. supra; Natoli et al., 1994, supra; Su and Schneider, 1996, supra). HBx was shown to stimulate RasGTP complex formation and to establish a cascade linking Ras, Raf, and MAP Kinase, which is essential for HBx activation of AP-1 (Benn and Schneider, 1994, supra) and NF-xcexaB (Su and Schneider, 1996, supra). However, the mechanism by which HBx stimulates RasGTP complex formation remains to be elucidated. Additional results have also shown that HBx stimulates cellular proliferation in quiescent cells and induces deregulation of cell cycle checkpoint controls in a Ras dependent manner (Benn and Schneider, 1995, Proc. Natl. Acad. Sci. USA 92:11215-11219), indicating that activation of Ras by HBx appears to a play a central role in defining HBx activities.
The present invention relates to the treatment and prevention of HBV infection by targeting activation of the Src family of kinases. The present invention also relates to compounds which inhibit HBx-mediated activation of the Src family of kinases as well as the downstream components of the Src kinase signaling cascade for the treatment of HBV infection.
The invention is based, in part on the Applicants"" surprising discovery that activation of a Src kinase signaling cascade is a critical function provided by HBx for mammalian hepadnavirus replication. The Applicants have shown that Src kinases are also activated during HBV infection of cultured cells and that this activation is an essential function of the viral HBx protein. Thus, the Applicants have demonstrated that the HBx-mediated activation of the Src kinase signaling cascade plays a fundamental role in mammalian hepadnavirus replication.
The Applicants have demonstrated that HBx mediated activation of Src kinase signaling cascade is an effective target for HBV anti-viral agents since activation of this pathway is essential for HBV replication. Therefore, targeting HBx for the treatment of HBV should result in a highly specific and efficacious method of blocking HBV replication. The Src family of kinases, although host cell gene products, are only activated in proliferating or differentiating cells, and in cells infected by many DNA and tumor viruses. Therefore, targeting the Src family of kinases for the treatment of HBV infection should result in a therapeutic with a high degree of efficacy and sufficient specificity with side effects no more toxic than chemotherapeutics currently used to treat cancer.
The present invention encompasses a variety of techniques and compounds to target the activities of HBx essential for HBV replication. In particular, these include, but are not limited to HBx-mediated activation of the Src kinase family signal transduction pathways for the treatment and prevention of HBV infection. The invention encompasses the use of known Src inhibitors to treat HBV infection. Examples of such specific inhibitors include, but not limited to: Src specific tyrosine kinase inhibitors, such as CsK, tyrphostin-derived inhibitors, derivatives of benzylidenemalonitrile, pyrazolopyrimidine PP1, and microbial agents, such as angelmicin B; and competitive inhibitors, such as small phosphotyrosine containing ligands. The invention also encompasses the use of known HBx inhibitors for the treatment of HBV, including, but not limited to, antisense RNAs directed to HBx. The present invention also relates to the use of inhibitors of downstream effectors of Src kinases, including but not limited to, cytoplasmic factors, such as Ras, Raf, focal adhesion kinase (FAK) and MAPK, and nuclear factors, such as Myc and cyclin-dependent kinases.
In another embodiment of the present invention gene therapy approaches, including dominant-negative mutants, antisense molecules and SELEX RNAs targeted to block Src kinase or HBx gene expression, may be used as a method to treat and prevent HBV infection and HCC. In yet another embodiment of the invention, upstream and downstream components and effectors of the Src kinase family signaling cascade may be targeted by gene therapy approaches to inhibit HBV infection.
The present invention further relates to screening assays to identify compounds which inhibit. HBx-mediated activation of the Src kinase signaling pathway and may be used to treat HBV infection and diseases and disorders associated with HBV infection.
The invention is illustrated by way of working examples which demonstrate that HBx mediates activation of a Src kinase signaling cascade and that activation of this signaling cascade is an essential function of HBx required to sustain HBV replication. The working examples of the present invention further demonstrate the ability of inhibitors of the Src kinase signaling cascade to inhibit HBV replication.
As used herein, the term xe2x80x9ctarget cellular genexe2x80x9d refers to those genes encoding members of the Src kinase family, including analogs and homologs of c-Src, Fyn, Yes and Lyn kinases and the hematopoietic-restricted kinases HcK, Fgr, LcK and Blk, and members of the Src kinase signaling pathway including both upstream and downstream components of the Src signaling cascade.
As used herein, the term xe2x80x9ctarget proteinxe2x80x9d refers to those proteins which correspond to Src kinase or members of the Src kinase family or components of the Src kinase signaling pathway or proteins encoded by the HBV genome, including HBx.
As used herein, the terms xe2x80x9cSrc kinasexe2x80x9d or xe2x80x9cSrc kinase familyxe2x80x9d refer to the related homologs or analogs belonging to the mammalian family of Src kinases, including, for example, the widely expressed c-Src, Fyn, Yes and Lyn kinases and the hematopoietic-restricted kinases Hck, Fgr, Lck and Blk.
As used herein, the terms xe2x80x9cSrc kinase signaling pathwayxe2x80x9d or xe2x80x9cSrc cascadexe2x80x9d refer to both the upstream and downstream components of the Src signaling cascade.
As used herein, the term xe2x80x9cto targetxe2x80x9d means to inhibit, block, or prevent gene expression, enzymatic activity, or interaction with other cellular or viral factors or contain a deletion or mutation in the catalytic or enzymatic portion of the target protein.
As used herein, the term xe2x80x9cdominant-negative mutantxe2x80x9d means those proteins or polypeptides which are functionally incompetent forms of the target protein and/or inhibit or modulate the enzymatic activity of the target protein or inhibit or modulate the interaction of the target protein with other cellular or viral factors.
As used herein, the term xe2x80x9ctreating or preventing HBV infectionxe2x80x9d means to inhibit the replication of the HBV virus, to inhibit HBV transmission, or to prevent HBV from establishing itself in its host, and to ameliorate or alleviate the symptoms of the disease caused by HBV infection. Treating or preventing HBV infection also encompasses inhibition of viral replication in cultured cells as well as animal hosts. The treatment is considered therapeutic if there is a reduction in viral load or viral pathogenesis, decrease in mortality and/or morbidity.
As used herein, the term xe2x80x9ctherapeutic agentxe2x80x9d refers to any molecule, compound or treatment, for example and antiviral, that assists in the treatment of a viral infection or the diseases caused thereby or an agent which alleviates or assists in the treatment of a viral infection or the diseases caused thereby or an agent which alleviates or assists in the treatment of disorders associated with HCC.
As used herein, the term xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d refers to a carrier medium that does not interfere with the effectiveness of the biological activity of the active ingredient, is chemically inert and is not toxic to the patient to whom it is administered.